In order to determine the kinetic parameters of glucose oxidation catalysed by the enzyme glucose oxidase (GO) the initial velocity of hydrogen peroxide formation was measured using a rotating disc electrode (RDE). The major advantage of this method is the possibility of continuous measurement of the increase in hydrogen peroxide concentration. This means that the real initial reaction rate V0 can be determined, which is required for constructing a double-reciprocal plot. Several combinations of substrate concentrations (glucose and oxygen) were used. The method, in which a platinum black RDE was used, appeared to be very useful. Product inhibition experiments showed that the ping-pong mechanism is valid for GO. The three kinetic parameters of this mechanism were determined by initial velocity experiments.